Year: 2025
Type: conference-abstract
Abstract: Understanding how new enzyme functions evolve, either on existing scaffolds, or completely de novo on previously non-catalytic scaffolds, is of great interest both from a fundamental biochemistry perspective, and from a biotechnological perspective. Several hypotheses have been put forward to rationalize enzyme evolution, one of which is that their conformational dynamics plays an important role in facilitating the emergence of new enzyme functions.1, 2 Such conformational dynamics, in turn, is an important feature that can be exploited in artificial enzyme design.3-5 In this talk, I will illustrate how we have engineered conformational dynamics to generate a de novo active site capable of catalyzing a non-natural reaction,6 with kcat/KM ∼ 5 × 103 M−1 s−1 and kcat ∼ 10 s−1. Subsequent computationally-focused ultra-low-throughput screening using FuncLib7 allowed us to obtain a Kemp eliminase with a catalytic efficiency (kcat/KM ∼2 × 104 M−1 s−1 and kcat ∼102 s−1) approaching that of naturally occurring enzymes.8 Finally, more recently, we have extended our design strategy to include computational stability design guided by NMR and simulation,9 and our latest Kemp eliminase from this strategy has catalytic efficiency (kcat/KM 4.3 × 105 M−1 s−1 and kcat ∼1700 s−1) exceeding the best Kemp eliminase designed to date using a proton abstraction mechanism,10 upon screening a small focused selection of variants. The broader implications of this for protein engineering will also be discussed.
Source: Structural Dynamics
DOI: 10.1063/4.0000840
Author: Lynn Kamerlin
Institution: Georgia Institute of Technology
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,200
OpenAlex ID: W7101443151